CN104357918A - Construction method of plasma free DNA library - Google Patents
Construction method of plasma free DNA library Download PDFInfo
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- CN104357918A CN104357918A CN201410690349.2A CN201410690349A CN104357918A CN 104357918 A CN104357918 A CN 104357918A CN 201410690349 A CN201410690349 A CN 201410690349A CN 104357918 A CN104357918 A CN 104357918A
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Abstract
The invention discloses a 'construction method for a small amount of DNA library'. The method comprises the following steps: carrying out end repairing on plasma free DNA or fragmented DNA and simultaneously carrying out phosphorylation modifying on 5' terminal to obtain modified DNA; later, implementing blunt end linking directly to an artificial linker and purifying to obtain relatively pure linker-containing DNA having a gap on joint; then repairing the gap by virtue of polymerase chain reaction (PCR) polymerase before PCR amplification to obtain repaired complete linker-containing DNA; and carrying out PCR amplification reaction and purifying a finished product to obtain a final high-throughput sequencing library. The method is not only simple, convenient and efficient, and is capable of avoiding a purification step and reducing DNA loss in a library construction process, but also can effectively solve a problem of linker self-linking which is easy to occur in a micro-DNA library construction process by virtue of a special linker and blunt end linking way; and meanwhile, by repairing the gap before amplification, sufficient complete templates are provided before amplification for the amplification reaction.
Description
Technical field
Repair the plasma dna library constructing method of breach before the present invention relates to amplification, particularly relate to free DNA library construction process in the pregnant woman blood plasma that can be applicable to without wound antenatal diagnosis.
Background technology
According to medical statistics, in the newborn infant of annual birth, about have 2 ~ 3% to suffer from congenital disorders or inborn defect, cause infant's confined to bed, disability for a long time, even die young, bring huge burden to society and family.Cause the reason of congenital disorders or inborn defect to mainly contain chromosome abnormalty, single gene inheritance disease, disease of multifactorial inheritance, teratogenesis tire factor etc., wherein chromosome abnormalty accounts for larger proportion.Stress the modern society of eugenic health, how to prevent and treat congenital disorders or inborn defect, become very important problem.And antenatal diagnosis, Timeliness coverage, in time termination of pregnancy are the important means of prevention hereditary defect infant birth.Traditional methods for prenatal diagnosis directly obtains fetal genetic material to diagnose by amniocentesis, fine hair biopsy and umbilical cord blood inspection, these antenatal diagnosis technology are all invasive, there is certain risk, fetal damage, parent may be caused to miscarry or intrauterine infection, even Intrauterine Fetal Death etc., to be difficult to by numerous pregnant woman and family accept.
The research such as Lo in 1997 reports in pregnant woman blood plasma the existence having fetus dissociative DNA, has research also to report afterwards and finds to there is fetal rna in pregnant woman blood plasma, and these great being found to be provide new possibility without wound antenatal diagnosis.3% ~ 30% of pregnant woman blood plasma STb gene is accounted in fetal cell-free DNA in maternal plasma, extraction maternal blood is just referred to without wound antenatal diagnosis, by carrying out high-flux sequence to maternal blood cell-free fetal DNA, detecting fetus by bioinformatic analysis and whether suffer from the numerical abnormalities of chromosomes diseases such as mongolism (mongolism), E trisomy (Edward's syndrome), 13-patau syndrome (handkerchief pottery Cotard), is an emerging technology in antenatal diagnosis field.Compared with invasive antenatal diagnosis, have that security is high, highly sensitive, specificity is good without wound antenatal diagnosis, detection time early, the feature such as quick.
At present, be generally after extracting the dissociative DNA in pregnant woman blood plasma without the library construction of plasma DNA in wound antenatal diagnosis process, by end reparation → purifying → adding the steps such as A → purifying → connecting joint → purifying → amplification → purifying has come.In library construction process, complex steps, through repeatedly purge process, need can cause the loss of DNA, there will be joint from connecting phenomenon, causing library construction efficiency low, of poor quality in the process simultaneously connected at joint.Or repaired by end and after adding A after purifying, connecting joint the step such as purifying, amplification, purifying come.Although decrease purification step, reduce the loss of DNA, still have joint to be difficult to solve from the problem connected.
Summary of the invention
The present invention aims to provide a kind of construction process of high-throughput sequencing library, builds storehouse to solve existing a small amount of DNA sample, and especially plasma DNA to be built in the process of storehouse DNA loss and easily occurred that joint is from the problem such as connecting.Present method is not only easy, efficient, decreases purification step, reduces the loss of DNA in library construction process, and uses the mode of connection of specific joint and flat end, effectively solves in minim DNA library construction process and easily occurs that joint is from the problem connected.Meanwhile, use special amplified library polysaccharase, before amplification, repair breach, before ensureing amplification, have enough complete templates to carry out amplified reaction.
A kind of a small amount of DNA library construction process, adopts following order, method steps:
(1) end is carried out to DNA fragmentation and repairs into flush end and 5 ' end phosphorylation, obtain repairing rear DNA,
(2) after repairing, DNA directly carries out blunt end cloning, purifying with joint, obtains that junction band is jaggy contains linker DNA;
(3) use PCR polysaccharase to carry out the repairing of breach, obtain after repairing complete containing linker DNA,
(4) pcr amplification, purifying, finally obtains high-throughput sequencing library.
Joint in described step (2) is that joint A and joint B, joint A are combined by SEQ ID NO1 and the annealing of SEQ ID NO2 equimolar amount, and described joint B is annealed by SEQ ID NO3 and SEQ ID NO4 equimolar amount and combines,
SEQ ID NO1：
5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
SEQ ID NO2：
5’-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATC-3’，
SEQ ID NO3：
5’-AGATCGGAAGAGCACACGTCTGAACTCCAGTCACIIIIIIATC-3’，
SEQ ID NO4：
5 '-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCT TCCGATCT-3 ', six bases " IIIIII " in described sequence 3 represent the sequence label in order-checking platform Multi-example mixing library.
Described order-checking platform is the platform that checks order for illumina, and described " IIIIII " can be the random combine of any six bases.
Described base sequence can be " ATCACG " in illumina checks order platform, or be " CGATGT ", or be " TTAGGC ", or be " TGACCA ", or be " ACAGTG ", or be " GCCAAT ", or be " CAGATC ", or be " ACTTGA ", or be " GATCAG ", or be " TAGCTT ", or be " GGCTAC ", or be " CTTGTA ".
Described step (1) uses T4 archaeal dna polymerase, e. coli dna polymerase I large fragment makes DNA repair into flat end.
Described step (1) uses T4 polynucleotide kinase to make the flat end 5 ' of DNA hold phosphorylation.
Described DNA fragmentation is plasma DNA, and size is 100-250bp; Or for genomic dna is 100-250bp through ultrasonication.
Library constructing method of the present invention, first carries out end to plasma DNA or fragmentation DNA and repairs into flush end, and carries out phosphorylation modification to 5 ' end simultaneously, obtains repairing rear DNA; Blunt end cloning is carried out in direct and manual splice afterwards, obtains junction band jaggy containing linker DNA; Purer junction band is obtained jaggy containing linker DNA after purified; Then before pcr amplification, use PCR polysaccharase to carry out the repairing of breach, complete containing linker DNA after obtaining repairing; Carry out pcr amplification reaction again, obtain amplified production; Final high-throughput sequencing library is obtained after purifying.
Library constructing method of the present invention, does not need to carry out purifying after carrying out the reparation of DNA end, utilizes reparation system directly to carry out the connection of joint, decreases the DNA loss that purification step causes; Library constructing method of the present invention, the joint of use only has one end to be flat end, ensure that be connected with target DNA in the right direction; The flat end of joint used, without phosphorylation modification, can not generate joint from connecting product when ensure that ligation; Library constructing method of the present invention, uses special archaeal dna polymerase to carry out the repairing of junction breach, carries out perfection reparation before ensureing pcr amplification to junction breach.
In high-flux sequence field, after library construction is good, usually need to carry out quality inspection to library.Quality inspection content comprises agarose gel electrophoresis assessment library fragments size; Adopting real-time fluorescence quantitative PCR assessment library concentration, whether having joint from connecting pollution.When library concentration is lower, follow-up high-flux sequence cannot be carried out.When containing the joint from connecting in library, high-flux sequence cannot be carried out equally.For Hiseq2000, it requires that library concentration is not less than 2nM, and does not pollute from the joint connected in library.
Technical scheme of the present invention, direct jointing after repairing end by employing, decreases purification step thus decreases DNA loss.The unidirectional flat end fitting used in the present invention does not carry out phosphorylation modification, thus avoids the joint that produces in library construction process from connecting phenomenon and ensureing the correct of joint closure.The PCR polysaccharase used in the present invention carries out the reaction of repairing breach before PCR reaction, ensure that enough complete templates carry out follow-up pcr amplification reaction.
Accompanying drawing explanation
Fig. 1 has shown library construction flow process of the present invention;
Fig. 2 has shown the electrophorogram of the embodiment of the present invention and reference examples library construction result;
Fig. 3 has shown embodiment of the present invention library real-time fluorescence quantitative PCR quality inspection melting curve figure;
Fig. 4 has shown reference examples library real-time fluorescence quantitative PCR quality inspection melting curve figure of the present invention;
Fig. 5 has shown the embodiment of the present invention and reference examples library real-time fluorescence quantitative PCR quality inspection melting curve comparison diagram.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
Carry out building according to the library construction flow process shown in Fig. 1.The joint sequence used is sequence listed in the claims 7.Wherein sequence 1 and sequence 2 equimolar amount are annealed and are combined into joint A, and sequence 3 and sequence 4 equimolar amount are annealed and be combined into joint B.
SEQ ID NO1：
5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
SEQ ID NO2：
5’-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATC-3’，
SEQ ID NO3：
5’-AGATCGGAAGAGCACACGTCTGAACTCCAGTCACIIIIIIATC-3’，
SEQ ID NO4：
5 '-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCT TCCGATCT-3 ', six bases " IIIIII " in described sequence 3 represent the sequence label in order-checking platform Multi-example mixing library.
Sample that embodiment uses is human normal plasma's dissociative DNA below.Commercial reagent box is used to carry out plasma DNA extracting.DNA uses Qubit photofluorometer to carry out quantitatively.
1, reference examples: plasma dna library construction
1) sample and reagent
Get Qubit quantitatively after plasma DNA 5ng, use commercial high-flux sequencing library to build test kit and carry out library construction.
2) end reparation carried out to plasma DNA and add dATP
Reaction system is as follows:
Reaction conditions: mix latter 20 DEG C and hatch 30 minutes.Hatch 30 minutes for 65 DEG C.
3) joint connects
Reaction system is as follows:
Reaction conditions: mix latter 20 DEG C and hatch 15 minutes.
Above-mentioned 3) system in add enzyme in the box of 3ul commercial reagent and carry out enzyme and cut.
Reaction conditions: mix latter 37 DEG C and hatch 15 minutes.
Magnetic beads for purifying is carried out to above-mentioned product.Operating process is carried out according to Ampure XP Beads purifying specification sheets.
4) pcr amplification
Reaction system is as follows:
Reaction conditions: 98 DEG C of denaturations 30 seconds; Then 98 DEG C of sex change 10 seconds, 65 DEG C 30 seconds, 72 DEG C 30 seconds, totally 10 circulations; Last 72 DEG C extend 5 minutes.
Magnetic beads for purifying is carried out to above-mentioned product.Operating process is carried out according to Ampure XP Beads purifying specification sheets.
To step 4) in PCR purified product to carry out Qubit quantitative.
2, embodiment: plasma dna library construction
1) sample and reagent
Get Qubit quantitatively after plasma DNA 5ng, use commercially available related reagent to carry out library construction.
2) end reparation and 5 ' end phosphorylation modification are carried out to plasma DNA
Reaction system is as follows:
DNA 25ul
Commercially available end repairs reaction mixture 10ul
Cumulative volume 35ul
Reaction conditions: mix latter 20 DEG C and hatch 30 minutes.Hatch for 70 DEG C and make enzyme deactivation in 15 minutes.
3) joint connects
Reaction system is as follows:
Reaction conditions: mix latter 20 DEG C and hatch 15 minutes.65 DEG C make enzyme deactivation in 20 minutes.
Magnetic beads for purifying is carried out to above-mentioned product.Operating process is carried out according to Ampure XP Beads purifying specification sheets.
4) breach and pcr amplification is repaired
Reaction system is as follows:
Reaction conditions: hatch 15 minutes for 72 DEG C, repairs breach; 95 DEG C of denaturations 3 minutes; 95 DEG C of sex change 15 seconds, 58 DEG C of annealing 15 seconds, 72 DEG C extend 30 seconds, totally 10 circulations; Last 72 DEG C extend 1 minute.
Magnetic beads for purifying is carried out to above-mentioned product.Operating process is carried out according to Ampure XP Beads purifying specification sheets.
To step 4) in PCR purified product to carry out Qubit quantitative.
3, detect: library quality inspection
1) 2% agarose gel is prepared.100ng is respectively got to the library of two in reference examples and embodiment and carries out agarose gel electrophoresis qualification.DNA marker uses TAKARA DL1000 marker 5ul.As shown in Figure 2, Fig. 2 is followed successively by TAKARA DL1000 marker, embodiment library, reference examples library to qualification result from left to right.As can be seen from Figure 2, embodiments of the invention library and reference examples library master tape (about 300bp) all more obvious.
2) by real-time fluorescence quantitative PCR detect further contrast library and experimental libraries whether have joint to pollute.1ul is respectively got in two libraries in reference examples and embodiment, uses the high-throughput sequencing library quantification kit of KAPA BIOSYSTEMS company to detect.As shown in Figure 3-Figure 5, Fig. 4 shows reference examples library to be had a little from connecting joint pollution (melting curve temperature 82 DEG C place) detected result, and embodiments of the invention library is polluted from connecting joint without any.
Can find out that the plasma DNA library of the method structure of above-described embodiment the application of the invention fundamentally avoids high-throughput sequencing library and builds the phenomenon easily occurring polluting from connecting joint, improves Library Quality from the above description.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention.All any amendments done within the spirit and principles in the present invention, equivalent replacement, improvement etc., be all included within protection scope of the present invention.
Claims (8)
1. a plasma DNA library constructing method, adopts following order, method steps:
(1) end is carried out to DNA fragmentation and repairs into flush end and 5 ' end phosphorylation, obtain repairing rear DNA,
(2) after repairing, DNA directly carries out blunt end cloning, purifying with joint, obtains that junction band is jaggy contains linker DNA;
(3) use PCR polysaccharase to carry out the repairing of breach, obtain after repairing complete containing linker DNA,
(4) pcr amplification, purifying, finally obtains high-throughput sequencing library.
2. construction process according to claim 1, joint in described step (2) is joint A and joint B, described joint A is annealed by SEQ ID NO1 and SEQ ID NO2 equimolar amount and combines, described joint B is annealed by SEQ ID NO3 and SEQ ID NO4 equimolar amount and combines
SEQ ID NO1：
5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’
SEQ ID NO2：
5’-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATC-3’，
SEQ ID NO3：
5’-AGATCGGAAGAGCACACGTCTGAACTCCAGTCACIIIIIIATC-3’，
SEQ ID NO4：
5 '-CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGTGCTCT TCCGATCT-3 ', six bases " IIIIII " in described sequence 3 are the sequence label in order-checking platform Multi-example mixing library.
3. construction process according to claim 2, described order-checking platform is the platform that checks order for illumina, and described " IIIIII " can be the random combine of any six bases.
4. construction process according to claim 3, described six base sequences are " ATCACG " in illumina checks order platform, or are " CGATGT ", or be " TTAGGC ", or be " TGACCA ", or be " ACAGTG ", or be " GCCAAT ", or be " CAGATC ", or be " ACTTGA ", or be " GATCAG ", or be " TAGCTT ", or be " GGCTAC ", or be " CTTGTA ".
5. construction process according to claim 1, described step (1) uses T4DNA polysaccharase, e. coli dna polymerase I large fragment makes DNA repair into flat end.
6. construction process according to claim 1, described step (1) uses T4 polynucleotide kinase to make the flat end 5 ' of DNA hold phosphorylation.
7. construction process according to claim 1, described PCR polysaccharase is commercially available archaeal dna polymerase.
8. construction process according to claim 1, described DNA fragmentation is plasma DNA, and size is 100-250bp; Or for genomic dna is 100-250bp through ultrasonication.
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